The extraction of intact biomolecules from a biological sample is an essential part of many laboratory and clinical diagnostic procedures. The instability of biomolecules such as nucleic acids, proteins, carbohydrates and lipids is well known and their integrity depends on a large number of parameters such as the physiological condition of the sample prior to removal from its original environment, how quickly the sample was removed from its source, the rate of sample cooling, sample storage temperature and the biomolecule purification method. It is well known that the treatment of the biological sample before and during biomolecule purification can lead to very important changes in the intactness and integrity of the sample analyte. For example it is well known that RNA in particular is an extremely labile molecule that becomes completely and irreversibly damaged within minutes if it is not handled correctly. Although RNA is perhaps one of the more labile biomolecules, proteins including post-translational modifications, lipids, small molecules of less than 2000 daltons that are essential to metabolic analysis and DNA can also be subject to substantial degradative processes.
Although endogenous cellular enzymes are responsible for the majority of degradative processes, the analyte will always tend to hydrolyse spontaneously during storage or processing and this process is largely dependent on the storage conditions such as temperature, water content, pH, light and stability and molecular weight of the analyte molecule but may also be dependent on the quality of the reagents used.
One of the most common and simple approaches for successful storage is to reduce the temperature of the biological sample. Generally samples are stored at temperatures below room temperature (20-24° C.); protein solutions at 4° C. or −20° C., nucleic acids in freezers at −20° C. or −80° C., in dry-ice or in liquid nitrogen. Anti-microbial agents such as sodium azide may be added to control microbial growth.
It is well known that RNA is particularly sensitive to degradation by enzymes, spontaneous hydrolysis, divalent metal cation catalysed hydrolysis, alkali sensitivity and cross-linking in FFPE (formalin-fixed paraffin-embedded) samples. Many metal solutions such as lead, magnesium and manganese are very destructive to RNA and are indeed essential for not only ribozyme but also nuclease activity such as DNase I, mung bean nuclease and S1 nuclease. Iron (2+) has been implicated in the oxidation of nucleobases as part of the Fenton reaction leading to translationally impaired rRNA and mRNA (Honda et al., (2005) J. Biol. Chem. 280, 20978-20986), for example the conversion of guanine to 8-oxo-guanine. Other possible catalytic roles of metal ions in enzymatic and nonenzymatic cleavages of phosphodiester bonds have been reviewed (Yarus, M. (1993) FASEB J. 7, 31-39). Indeed chelators such as EDTA and EGTA are frequently added to RNA or RNA lysis solutions for the purpose of reducing RNA degradation by removing metal ions. Ribonucleases (“RNases”) are a large group of ubiquitous enzymes associated with many sources including microbes, human skin, dust and the content of cells and tissues. They are also readily released from intra-cellular vesicles during freeze-thawing. Certain tissues including the pancreas are known to be particularly rich in RNase A. RNase A is one of the most stable enzymes known, readily regaining its enzymatic activity following, for example, chaotropic salt denaturation making it extremely difficult to destroy. A high concentration of chaotrope such as guanidine (4-6M) is required to destroy RNase activity (Thompson. J. and Gillespie. D. Anal Biochem. (1987) 163:281-91).
There are several methods for inhibiting the activity of RNases such as using; (i) ribonuclease peptide inhibitors (“RNasin”) an expensive reagent only available in small amounts and specific for RNase A, B and C, (ii) reducing agents such as dithiothreitol and β-mercaptoethanol which disrupt disulphide bonds in the RNase enzyme, but the effect is limited and temporary as well as being toxic and volatile, (iii) proteases such as proteinase K to digest the RNases, but the transport of proteinases in kits and their generally slow action allows the analyte biomolecules to degrade, (iv) reducing the temperature to below the enzyme's active temperature; commonly tissue and cellular samples are stored at −80° C. or in liquid nitrogen, (v) anti-RNase antibodies, (vi) precipitation of the cellular proteins including RNases, DNA and RNA using solvents such as acetone or kosmotropic salts such as ammonium sulphate, a commercialised preparation of ammonium sulphate is known as RNAlater™ (Sigma-Aldrich, USA; LifeTechnologies, USA; Qiagen, Germany), (vii) detergents to stabilise nucleic acids in whole blood such as that found in the PAXgene™ DNA and RNA extraction kit (PreAnalytix, Germany), (viii) chaotropic salts, (ix) alcohols such as those found in the PAXgene™ Tissue stabilisation reagent (PreAnalytix, Germany). A range of such chaotropic mixtures are set out in RNA Isolation and Analysis, Editor. Jones (1994) Chapter 2.
The primary goal of sample storage is to minimise any changes to the analyte biomolecule that may be introduced as a result of the pre-analytical procedure and sample purification so that the analytical result resembles as closely as possible the original in vivo complexity and diversity of the biomolecules, thereby improving assay accuracy, sensitivity and specificity. Whilst there are various methods and products that are available to reduce pre-analytical variation, all suffer from various drawbacks making their use problematic or sub-optimal. Procedures that are effective at stabilising one class of biomolecules are often ineffective at stabilising others so that the technician is obliged to choose a specialised reagent and procedure for each biomolecule analyte. For example the PAXgene™ system (PreAnalytix) (U.S. Pat. Nos. 6,602,718 and 6,617,170) can be used for nucleic acids but not proteins and requires lengthy purification steps with multiple wash buffers, whilst cocktails of protease inhibitors help to protect proteins from degradation but not nucleic acids. The PAXgene™ tissue stabilisation kit requires two separate treatments of the tissue and involves toxic and flammable chemicals. RNAlater™ treatment of tissues reduces their utility for immunohistochemistry, histology and increases tissue hardness without fully protecting the RNA, nevertheless it has been adopted as the gold standard for RNA preservation.
It is not always possible to purify RNA at the time or site where the sample is extracted, for example a biopsy from a hospital operating theatre or a blood sample from a doctor's office. In these cases, the sample must be very carefully stored prior to RNA extraction, which might be carried out within as little as 30 minutes but would more commonly occur only after several hours or days following processing by the hospital pathology laboratory. Often the time and temperature of the pre-analytical step is poorly recorded leading to ambiguous knowledge of the quality of the sample. As a consequence, it has been necessary to develop separate sample storage conditions for each type of tissue and final use of the RNA. As already stated this generally involves using either a dedicated stabilisation solution such as RNAlater or PAXgene or immediately freezing the sample in liquid nitrogen. At least in the case of the PAXgene stabilisation reagent, incomplete removal of the stabiliser will negatively impact RNA yields during purification (PAXgene Blood RNA Kit Handbook, June 2005).
Tissue storage may be effected by tissue fixation using a fixative. ‘Fixation’ refers to increasing the mechanical strength, hardening, preserving and increasing stability of the treated biological sample such as fresh cells, biopsy or tissue, and maintains the sample in a state as similar as possible to that of the original fresh sample in situ, in its natural state. Fixation is commonly used in pathology, histology, histochemistry, cytochemistry, anatomical studies and studying cells, and generally precedes additional steps such as storage, embedding, staining, immunohistochemistry and/or immunocytochemistry. The process of fixation ideally inhibits enzymes such as nucleases and proteases, stops microbial growth on the sample and maintains both gross tissue morphology as well as cellular ultrastructure such as golgi, nucleus, endoplasmic reticulum, mitochondria, lysosomes and cytoplasmic membranes. As one example, the preservation of the correct cell morphology is important for a pathologist to diagnose the presence, type and grade of cancer in a patient, but in order to do this correctly the sample must also be capable of becoming correctly stained or labelled with antibodies for immunohistochemistry. Commonly the sample is treated with a 1-5% aqueous buffered solution of formalin (formaldehyde) paraformaldehyde or glutaldehyde for 1-24 hours at room temperature in order to allow cross linking of proteins and other cellular components and then, following tissue sectioning, stained with Haematoxylin and Eosin stain (H&E). Although glutaraldehyde can also be used its rate of penetration into the tissue is slower than with formaldehyde (which penetrates at approximately 1 mm per hour when 18-20 volumes are added relative to the tissue volume). Whilst RNA can also be preserved in this manner, in general it becomes highly degraded during or after formalin fixation making gene expression analysis highly problematic and artifactual. One specific problem is that the RNA analyte becomes cross-linked with other biomolecules such as proteins so that they subsequently need to be released prior to analysis, this process is generally very harsh requiring extended periods at elevated temperatures which leads to significant RNA degradation. Another problem of fixation is maintaining soluble analytes such as RNA and proteins in the cell so they can be integrated by for example, in situ hybridization or immunohistochemistry. Yet another problem with formalin fixation in particular is that the covalent modification of the cellular proteins results in the loss of antigenic immunorecognition which can render immunohistochemistry techniques difficult or impossible depending on the antibody. As one further example, formalin fixed tissues are routinely embedded in paraffin wax to allow the tissue block to be thinly sliced and examined microscopically (FFPE). Other common tissue fixation methods involve using methanol, ethanol or acetone that result in protein precipitation rather than cross-linking. Methods to fix tissues, maintain good RNA quality whilst allowing immunohistochemical staining are particularly needed. It is well known that commonly used fixatives such as formaldehyde, paraformaldehyde, gluataldehyde and methanol are highly toxic potential carcinogens, whilst ethanol and acetone are highly flammable. The ideal fixative should work on a wide variety of tissues including neural, lymphoid and fatty, preserve large pieces of tissues, and be compatible with immunohistochemical, histochemical and in situ hybridisation and other specialised techniques. It should also be compatible with automated tissue fixation procedures. A review with detailed protocols has been published by Bancroft (2008) ‘Theory and Practice of Histological Techniques’ and by Stanta (2011) ‘Guidelines for Molecular Analysis in Archive Tissues’ whilst representative examples of fixed and stained tissues can be found in Ross and Pawlina (2011) ‘Histology A Text and Atlas’.